Project Summary/Abstract The majority of vaccine-elicited antibody responses to influenza are dominated by off-target, immunodominant, and non-neutralizing activities. However, recent work indicates that human B cell receptors (BCRs) containing the antibody VH gene IGHV1-69 possess V gene-encoded specificity for a functionally conserved site of vulnerability, the stem-epitope of the influenza glycoprotein hemagglutinin (HA), a target of broadly neutralizing antibody (bnAb) responses. To experimentally evaluate IGHV1-69 stem-epitope targeting as a gene-encoded template for building a universal influenza vaccine, our lab has generated transgenic mice in which antibody development proceeds via normal human VDJ recombination, but is constrained to a single V gene, IGHV1-69. My preliminary data indicates that IGHV1-69 usage in itself is sufficient to refocus the antibody response to the HA stem epitope and is protective from an unmatched viral challenge. This is a major paradigm shift in rational vaccine design, namely that broad protection may be generated through activation and amplification of gene- encoded antibody responses. I now propose to assess the breadth of the IGHV1-69 protective response as well as examine if this encoded targeting activity is regulated by a single amino acid change, present in 15% of the global population, using an IGHV1-69 SNP constrained mouse model. Additionally, to define a means for clinical development, I have applied a RNA bacteriophage platform for peptide display and affinity selection to derive multivalent virus like particles (VLPs) that specifically engage IGHV1-69 germline BCRs, allowing for selective expansion of IGHV1-69 precursors from the full human immunoglobulin repertoire. I propose to evaluate selective IGHV1-69 priming in two models: 1). C57BL/6 mice adoptively transferred with IGHV1-69 B cells, and 2). The Trianni mouse, the latest industry-standard humanized vaccine model. Priming will then be followed by boosting with HA immunogens to amplify HA stem-epitope targeting antibody responses. This study will define a genetically encoded basis for bnAb elicitation and aims to overcome the failure of traditional approaches to influenza vaccination.